Data_Sheet_1_Accumulation of Target Gene Mutations Confers Multiple Resistance to ALS, ACCase, and EPSPS Inhibitors in Lolium Species in Chile.DOCX

Different Lolium species, common weeds in cereal fields and fruit orchards in Chile, were reported showing isolated resistance to the acetyl CoA carboxylase (ACCase), acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibiting herbicides in the late 1990s. The first case of multiple resistance to these herbicides was Lolium multiflorum found in spring barley in 2007. We hypothesized that other Lolium species may have evolved multiple resistance. In this study, we characterized the multiple resistance to glyphosate, diclofop-methyl and iodosulfuron-methyl-sodium in Lolium rigidum, Lolium perenne and Lolium multiflorum resistant (R) populations from Chile collected in cereal fields. Lolium spp. populations were confirmed by AFLP analysis to be L. rigidum, L. perenne and L. multiflorum. Dose-response assays confirmed multiple resistance to glyphosate, diclofop-methyl and iodosulfuron methyl-sodium in the three species. Enzyme activity assays (ACCase, ALS and EPSPS) suggested that the multiple resistance of the three Lolium spp. was caused by target site mechanisms, except the resistance to iodosulfuron in the R L. perenne population. The target site genes sequencing revealed that the R L. multiflorum population presented the Pro-106-Ser/Ala (EPSPS), Ile-2041-Asn++Asp-2078-Gly (ACCase), and Trp-574-Leu (ALS) mutations; and the R L. rigidum population had the Pro-106-Ser (EPSPS), Ile-1781-Leu+Asp-2078-Gly (ACCase) and Pro-197-Ser/Gln+Trp-574-Leu (ALS) mutations. Alternatively, the R L. perenne population showed only the Asp-2078-Gly (ACCase) mutation, while glyphosate resistance could be due to EPSPS gene amplification (no mutations but high basal enzyme activity), whereas iodosulfuron resistance presumably could involve non-target site resistance (NTSR) mechanisms. These results support that the accumulation of target site mutations confers multiple resistance to the ACCase, ALS and EPSPS inhibitors in L. multiflorum and L. rigidum from Chile, while in L. perenne, both target and NTSR could be present. Multiple resistance to three herbicide groups in three different species of the genus Lolium in South America represents a significant management challenge.

智利麦田与果园中的常见杂草——黑麦草属（Lolium）不同物种，于20世纪90年代末被报道对乙酰辅酶A羧化酶（acetyl CoA carboxylase, ACCase）、乙酰乳酸合酶（acetolactate synthase, ALS）及5-烯醇式丙酮酰莽草酸-3-磷酸合酶（5-enolpyruvylshikimate-3-phosphate synthase, EPSPS）抑制剂类除草剂产生单抗性。
2007年，研究者在春大麦田中发现的多花黑麦草（Lolium multiflorum）是这类除草剂多重抗性的首例报道。
本研究推测，其他黑麦草属物种或许也进化出了多重抗性。
本研究针对从智利麦田采集的硬黑麦草（Lolium rigidum）、多年生黑麦草（Lolium perenne）及多花黑麦草（Lolium multiflorum）抗性（Resistant, R）种群，对其针对草甘膦（glyphosate）、禾草灵甲酯（diclofop-methyl）与碘甲磺隆钠盐（iodosulfuron-methyl-sodium）的多重抗性特征进行了表征。
通过扩增片段长度多态性（Amplified Fragment Length Polymorphism, AFLP）分析，确认供试黑麦草属种群分别为硬黑麦草、多年生黑麦草与多花黑麦草。
剂量反应试验证实，上述三个黑麦草属物种均对草甘膦、禾草灵甲酯及碘甲磺隆钠盐表现出多重抗性。
酶活性测定（ACCase、ALS与EPSPS）结果显示，除多年生黑麦草抗性种群对碘甲磺隆的抗性外，三个黑麦草属物种的多重抗性均由靶标位点机制介导。
靶标位点基因测序结果显示：多花黑麦草抗性种群存在EPSPS基因Pro-106-Ser/Ala突变、ACCase基因Ile-2041-Asn与Asp-2078-Gly复合突变，以及ALS基因Trp-574-Leu突变；硬黑麦草抗性种群则存在EPSPS基因Pro-106-Ser突变、ACCase基因Ile-1781-Leu与Asp-2078-Gly复合突变，以及ALS基因Pro-197-Ser/Gln与Trp-574-Leu复合突变。
而多年生黑麦草抗性种群仅存在ACCase基因Asp-2078-Gly突变；其草甘膦抗性可能源于EPSPS基因扩增（无碱基突变但基础酶活性较高），而碘甲磺隆抗性则可能涉及非靶标抗性（non-target site resistance, NTSR）机制。
上述结果表明，智利地区的多花黑麦草与硬黑麦草通过积累靶标位点突变，获得了对ACCase、ALS及EPSPS抑制剂类除草剂的多重抗性；而多年生黑麦草则同时存在靶标抗性与非靶标抗性两种机制。
在南美洲的三个黑麦草属物种中同时出现对三类除草剂的多重抗性，这对杂草综合防控工作构成了严峻挑战。